Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade
Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade
复制标题
DOI：
10.1074/jbc.m110.125310
复制
发表时间：
2011-01-07
影响因子：
4.8
通讯作者：
Diamandis, Eleftherios P.
中科院分区：
文献类型：
--
作者：
Eissa, Azza;Amodeo, Vanessa;Diamandis, Eleftherios P.
激肽释放酶相关肽酶 8 (KLK8) 是一种相对未知的表皮蛋白酶。尽管KLK8被提议调节皮肤屏障的剥落和恢复，但其催化活性从未在人类表皮中得到证实，其调节因子和靶点仍然未知。在此，我们首次阐明了人类非掌跖角质层和汗液中 KLK8 的离体活性。大多数角质层和汗液中的 KLK8 具有催化活性，在 pH 8.5 时表现出最佳活性，在 pH 5 时表现出相当大的活性。我们还表明，KLK8 是一种角质形成细胞特异性蛋白酶，不由人类黑素细胞或真皮成纤维细胞分泌。钙诱导终末角质形成细胞分化后，KLK8 分泌显着增加，表明这种蛋白酶在上表皮中发挥着积极作用。使用巴斯德毕赤酵母中产生的前 KLK8 和成熟 KLK8 重组蛋白，通过体外动力学测定鉴定了 KLK8 活性的潜在激活剂、调节剂和靶标。成熟的 KLK8 活性可被钙离子和镁离子增强，并被锌离子和 Arg(164) 后的自动裂解减弱。在筛选 FRET 淬灭肽文库的 KLK8 裂解后，观察到胰蛋白酶样特异性，在 P1-P1'-P2' 处对 (R/K)(S/T)(A/V) 具有最高偏好。我们还证明，KLK5 和赖氨酰内肽酶激活潜在的 pro-KLK8，而活性 KLK8 在体外靶向激活 pro-KLK11、pro-KLK1 和 LL-37 抗菌肽。总之，我们的数据确定 KLK8 是人类角质层和汗液中的一种新的活性丝氨酸蛋白酶，我们提出了增强其参与皮肤屏障蛋白水解级联的调节剂和靶点。 KLK8 升高和过度活跃对脱屑性和炎症性皮肤病的影响仍有待研究。
Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg(164). Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1'-P2'. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.